Anaiytzca Chumca Acta, 275 (1993) 23-48 23 Elsevler hence Publishers B V , Amsterdam
Metabolism of anabolic steroids in man: synthesis and use of reference substances for identification of anabolic steroid metabolites
Will1 Schanxer and Manfred Domke LkutscheS’rthochschuk Koln, Inrhtut f?u Bwchemre, Carl-L&m-Weg 4 5ooO Cologne 41 (Germuny) (Recewed 20th May 1992)
AhStl-&
The use of anabohc steroids was banned by the International Olympic Comnuttee for the first tune at the Olympic Games m Montreal m 1976 Since that tie the nususe of anabohc steroxls by athletes has been controlled by analysis of urme extracts by gas chromatography-mass spectrometry @C-MS) The excreted steroids or their metabohtes, or both, are isolated from urme by XAD-2 adsorption, enzymatx hydrolyss of coqlugated excreted metabobtes anth /?-glucuromdase from Es&en&u co& bqmd-bqmd extractlon mth diethyl ether, and courted mto tnmethylsdyl (‘INiS) derwatwes The confirmation of an anabobc stenxd nususe ts based on comparison of the electron nnpact lomzation (EI) mass spectrum and GC retention tie of the isolated steroid and/or its metabohte with the El mass spectrum and GC retention time of authentic reference substances For this purpose excretion studies with the most common anabobc steroids were performed and the mam excreted metabobtes were synthesued for bolasterone, boldenone, 4-chiorodehydromethyltestosterone, clostebol, drostanolone, tluoxymesterone, forme- bolone, me&a&one, mesterolone, metandlenone, methandnol, metenolone, methyltestosterone, nandrolone, norethandrolone, oxandrolone and stanozolol The metabobsm of anabohc Hero& the synthesis of theu mam metabohtes, theu GC retentmn and EI mass spectra as TMS dewames are dIscussed &ywor& Gas chromatography, Gas chromatography-mass spectrometv, Anabobc steroids, Metabohsm
The misuse of testosterone and anabohc abohc steroids and then metabohtes are isolated steroids by athletes to improve their performance from urme, derrvatrzed wrth N-methyl-N-tnmeth- has led to a ban on anabohc steroids by the ylstlyltnfluoroacetarmde (MSTPA) and analysed International Olympic Comnnttee (IOC) and na- by gas chromatography (GC) and mass spectrom- tional and mtematronal sports federations An- etry (MS) usmg electron impact (ED romxatron at abohc steroids were banned for the first time at 70 eV Because most anabohc steroids are com- the Olympic Games 1976 m Montreal and testos- pletely metabohxed and no parent steroid is ex- terone at the Olympic Games 1984 m Los Ange- creted, the metabolism of the anabohc steroids les To control the misuse of an anabohc steroid must be known Papers on the metabolism of the urmes of athletes are collected directly after anabohc sterords m man were available for bolde- competrtron or out of competrtron and have to be none [1],4-chlorodehydromethyltestosterone [2,3], analysed by IOC accredited laboratories The an- metandrenone [4,5], methyltestosterone [6], nan- drolone [71 and norethandrolone [8] The identifi- catron of these metabohtes was based on the Comzvpondence to W Schanzer, Deutsche Sporthochschule Koln, Instltut fir Bmchenue, Carl-Diem-Weg 6,5ooO Cologne isolation and purrficatron of urmary excreted 41 (Germany) metabohtes after application of large amounts of
0003~2670/93/$06 00 Q 1993 - Elsevler Science Publishers B V All nghts reserved 24 W Schanzer and M Lkwukz/Am! Chm Acta 275 (1993) 23-48 the anabohc steroid and characterlzatlon by melt- mmmm hydnde, chromnun tnoxlde, platmum mg points and mfrared (IR), ‘H nuclear magnetic dloxlde and 10% palladium on charcoal were resonance (NMR) and mass spectrometry At purchased from Aldrich (Stemhelm) Bolasterone least the proposed structure of the metabohte was purchased from UpJohn, Cchlorodehydro- was elucidated by its synthesis methyltestosterone was a gift from Jenapharm GC with fused-s&a capdlary columns corn- (Jena), fluoxymesterone was a gift from Cuba- bmed with MS with EI lomzatlon allows very fast Geigy, furazabol was a g& from Danchl (Tokyo), and relatively simple sample preparation to iso- oxandrolone was a gti from Searle, oxymesterone late small amounts of anabolrc steroids and their was a @ft from Carlo Erba, mesterolone, metabohtes from urine [9-113 The isolated metenolone and metenolone acetate were g&s metabohtes are characterized by then GC reten- from Schermg (Berlin), formebolone was a gift tion times and EI mass spectra A metabohte is from A H Beckett (London), drostanolone propl- identified when the companslon wth an authen- onate was a gift from Grunenthal (Aachen), tic reference substance shows the same GC reten- boldenone, clostebol acetate, mestanolone, tion time and EI mass spectrum metandlenone, methandnol, methandrlol dlpro- As most of the anabohc steroid metabohtes plonate, methyltestosterone, nandrolone and were not commercially avalable m earlier times, stanozolol were purchased from Sigma (Delsen- so-called “reference substances” were obtained dorf), norethandrolone, 6&hydroxytestosterone from urme m excretion studies vvlth anabohc and 6/3-hydroxyandrostenedlone were purchased steroids A cntlclsm of this method was the low from Steralolds via Paesel (Frankfurt) accuracy of a urinary “reference substance” for N-methyl-N-trlmethylsllyltrlfluoroacetamlde which the structure 1s not exactly known and the (MSTFA) was synthesued m the laboratory All possible nnpur?ty of the urmary extract other reagents and solvents were of analytlcal-re- To circumvent this disadvantage we started m agent grade 1987 the synthesis of the mam metabohtes of those anabohc steroids which were mainly mls- Metabohsm studws used m sports The synthesis of metabohtes of Metabohsm studies were performed by oral boldenone [ 121,metandlenone [ 131,stanozolol [ 141 application of the anabohc steroids I-XX to male and the synthesis of 17-epunenc steroids [151 volunteers and by self-admmlstratlon Usually a have been published Further pubhcatlons are m smgle dose of 20 mg of steroid was applied and preparation the followmg urine samples were collected and This paper gives an oveMew of the mam stored at 4°C metabolic pathways, the synthesis of the mam metabolrtes of anabohc steroids and their tnmethylsllyl (TMS) derlvatlzatlon for GC-MS Isolatwn of an&&c steroids and theu metabo- and presents the GC retention mdlces and EI htes mass spectra of the anabohc steroids excreted The steroids and/or then metabohtes were unchanged and their mam metabohtes, which are isolated as described by Domke et al [9] wth routmely used for screemng and confirmation some modifications The actual sample prepara- The pharmacokmetics of the excreted steroids tion 1s described and metabohtes will not be discussed Isolatwn of u?aconJugatedexcreted stero& To 5 ml of urme are added 0 5 g of sodium EXPERIMENTAL hydrogencarbonate-potassium carbonate (2 1, w/w> solid buffer and 50 ng of 4a-hydroxy- Steroids and chermcals stanozolol as mtemal standard and the uncon~u- K-Selektrlde (1 M potassmm tn-set -butyl- gated steroids are extracted with 5 ml of dlethyl borohydnde m tetrahydrofuran), hthmm alu- ether (dlstllled over calcium hydride) The ether W SchamerandM Donke/AnalChm Acta275(1993)23-48 25 layer IS transferred after centrrfugatlon and evap- Column C was used (see GC-FID parameters) orated to dryness under vacuum The column temperature was programmed from See also the lsolatlon of conlugated steroids 2ooOC at 4O”C/mm to 320°C (held for 3 mm) The mjector temperature was 300°C and the interface Isolatwn of conlugated excreted sterouii and temperature 3u)“C metabohtes ConJugated fractwn The instruments and car- To 2 ml of urme 1s added 1 pg of methyl- rier gas were the same as above Column A was testosterone (mternal standard) and the steroids used (see GC-FID parameters) The column are adsorbed on Amberhte XAD-2 polystyrene temperature was programmed from 180°C at resin The XAD-2 columns (Pasteur pipette, CC/mm to 240°C and at 15”C/mm to 320°C closed with a glass pearl, bed height 2 cm) are The injector temperature was 300°C and the m- washed with 2 ml of doubly dlstllled water and terface temperature 320°C eluted wth 2 ml of methanol The methanohc eluate 1s evaporated to dryness and the residue 1s GC-FID parameters dissolved m 1 ml of 0 2 M sodium phosphate An HP 5880 gas chromatograph was used The buffer (pH 7) carrier gas was hehum at 15 ml/mm at 18o”C, At this stage a separation of conjugated and splitting ratio 1: 10, pressure 27 PSI The column unconjugated excreted steroids 1s possible The temperature was programmed from 180°C at unc.onJugated steroids can be extracted with 5 ml 5”C/mm to 320°C Column A was Hewlett- of dlethyl ether The remammg ether must be Packard Ultra-l fused s&a, cross-linked methyl- removed by evaporation of the buffer solution for silicone (OV-11, 17 m X 0 2 mm 1 d , film thrck- a short time To the buffer solution 50 ~1 of ness 0 11 pm Columu B was Hewlett-Packard P-glucoromdase from E cob (K12, Boehrmger, HP-1 fused slhca, cross-linked methyls&cone Mannheun) are added and after 1 h at 50°C the (OV-0, 17 m x 0 25 mm I d , film thickness 0 33 buffer solution 1s made alkalme (pH 9-10) with pm Column C was Chrompack WCOT fused 250 ~1 of 5% potassmm carbonate solution and slhca CP-SIL 8CB, cross-lmked 5% phenyl- the steroids are extracted mth 5 ml of dlethyl methyls&one (SE-54), 17 m x 0 25 mm 1 d , film ether on a vlbro mixer for 30 s After centrtiga- thickness 0 25 pm Column D was Macherey- tlon the ether layer 1s transferred and evaporated Nagel Permabond, fused silica, cross-lmked 5% to dryness under vacuum phenyl-methylsdlcone (SE-541, 17 m x 0 25 mm 1 d, film thickness 025 pm Column E was Denvatlzatwn for GC-MS analyszs Hewlett-Packard HP-5, fused slllca, 5% phenyl- UnconJugated fractzon The dry residue IS methyl&cone @E-54), 17 m X 0 2 mm 1 d , film derlvatlzed with 50 ~1 of N-methyl-N-&methyl- thlckuess 0 33 pm sdyltrffluoroacetamlde-mudazole (MSTFA-Inu) (100 2, v/w) and heated for 30 mm at 80°C [91 Synthesu of reference substances (general de- ConJugated fractwn The dry residue is deriva- scnptwn) tLzed with 100 ~1 of MSTFA-ammonmm &datwn of secondary hydroxy groups mth Iodide-dlthloerythntol (1000 2 4, v/w/w) and chromrum trwxuk The substance which was to be heated for 15 mm at 60°C This reaction murture oxldlzed was dissolved m 96% acetic acid and 1s eqmvalent to a nuxture of MSTFA-tnmethyl- with stlrrmg a solution of 10% chrommm trloxlde lodosllane (TMIS) (1000 2, v/v) [161 m 96% acetic acid was added wthm 10 mm In general an equunolar addition of chrommm trlox- CC-MS parameters lde was sufficient, yleldmg quantltatlve oxldatlon UnconJugated fractwn A Hewlett-Packard HP After 30 mm of reaction the reaction rmxture was 5890 gas chromatograph and an HP 5970 mass neutrahzed and then made alkalme (pH 12-14) spectrometer were used. The tamer gas was he- wth a cooled aqueous solution of 20% potassmm hum at 1 ml/mm at 18O”C, sphttmg ratro 1 10 hydroxide The reaction products were extracted W Schanzer ad M Dontkc /Ad Chm Acta 275 (1993) 23-48 wrth a tenfold volume of dtethyl ether The ether dissolved m ethanol or ~sopropanol. The ethano- layer was washed wrth water, drted over sodmm hc solution was exposed to sunhght whrle sturmg sulphate and evaporated to dryness under vac- for about 4-8 h The androsta-3,5-dren-3-enol UUIU and androsta-1,3,5-trren-3-enol TMS-ethers were Reductwn of the 4,5-double bond Reductron of autooxrdrxed under these condrtrons at C-6, yreld- the 4,5-double bond m 3-keto-4-ene steroids was mg a 6/3-hydroxyandrost-4-en-3-one or 6/3-hy- performed wtth hydrogen usmg methanol or droxyandrosta-1,4-dren-3-one structure Durmg methanol-6 M potassmm or sodium hydroxtde further sample preparatron remammg TMS (10 1, v/v> as solvent and platmum dtoxtde or groups, e g , 17/3-0-TMS, were hydrolysed A de- 10% palladmm on charcoal as catalyst tatled descrrptron of thts syntheses ts m prepara- Reductwn of 3-k&o groups wzth bthuun alu- tion. muuum hydrzde Reductron with hthmm alu- munum hydride was performed 111absolute dry dtethyl ether or m tetrahydrofuran (both freshly RESUJaTS AND DISCUSSION dtstrlled over calcmm hydrrde) dependmg on the solubrltty of the substance which was to be re- Syntheszs of reference substances duced The reaction mtxture was held under ar- A general descrrptron of the often used reac- gon and whtle sttrrmg solid hthmm alummmm tion schemes for the synthesis of anabohc steroid hydride was added (1 equnnolar excess) After a metabohtes will be descrrbed The syntheses of further 15 mm the reaction rmxtclre was poured boldenone [ 121, comugated metandtenone [13], mto the same volume of water and the reaction stanozolol [14] metabohtes and 17-epmertxatron products were extracted wrth a tenfold volume of of 17a-methyl-17&hydroxy sterords [U] have al- drethyl ether The ether layer was washed wrth ready been pubhshed The synthesis of other water, drred over sodmm sulphate and then evap- metabohtes wrll be pubhshed elsewhere. orated to dryness under vacuum All the synthesized metabohtes were obtamed Reductwn of 3-keto groups wrth K-Select&e as pure crystals wtth a yteld between 60 and 2000 The reactron and sample preparatron were per- formed m the same way as the reaction wrth mg hthmm ahunmmm hydrrde. The K-Selektrrde was added m an equnnolar amount to the sttrred &uiatwn of secondary hydmxy groups wuh solutron held under argon wtthm 10 mm chnmuum trwxuie Reductwn of 3-k&o groups wtth hydrogen The A selectrve oxrdatron of only the 3-hydroxy reduction of 3-keto groups wrth hydrogen usmg group or 17&hydrow group when both hydroxy platinum dtoxtde or 10% palladmm on charcoal groups were present was not posstble As all was performed m the same way as the reduction synthesrxed metabohtes have a 17-keto- or 17-hy- of the 4,5-double bond, but the reaction was droxy-17-methyl group, thrs unspecttIc oxrdatron prolonged to 3 days was chosen followed by a group-selective reduc- 6/3-Hydroxylatwn. Stereospecrfrc 6/3-hydroxyl- tron of the 3-keto group only atron of androst-4en3-ones and androsta-l,Cdr- ene3-ones was performed wtth the followmg pro- Reductwn of the 4,Pdouble bond cedure The substance was dissolved m ethyl ac- The reductton performed 111methanol yrelded etate-MSTFA-ammomum iodide (100 10 0 5, m general a mrxture of 5a- and Sj?-isomers m v/v/w) and refluxed for 15 mm The obtamed comparable amounts whereas the reaction m androsta-3,5-dren-3-enol and androsta-1,3,5- methanol-aqueous potassmm hydroxrde or sodr- trren3-enol TMS-ethers were extracted with n- urn hydroxrde favours the productron of the 5p- pentane after addition of potassmm carbonate isomer To obtam a Scr-isomer methanol was cho- and water to the reaction mrxture The n-pentane sen as solvent, yreldmg 5a- and S/3-isomers, whrch phase was evaporated to dryness and the restdue were then separated by column chromatography W Schanzerand M Do&e/Ad Chim. Acta 275 (1993) 23-48 27 on s&a gel followed by repeated crystalllzatlon IS present, e g , m 5a-estrane-3,17-dlone, both The commonly used Birch reaction which yields keto groups will react munedlately wth the mamly the Sa-Isomer was not apphed reagent The reductron IS stereospecfic to 3-keto groups which til be reduced mamly to 3&hy- Reductwn of 3-keto groups wrth hthuun alu- droxy (Table 1) when the A-rmg has a 5a or muuum hydnde 4-ene configuratlon but the 3-keto group ~11 be Reduction wth hthmm alummmm hydnde 1s reduced m over 80% yield to 3a-hydroxy when not group selective If more than one keto group the A-ring has a S/3 configuration (Table 1)
TABLE 1 Reduction yields of 3-keto groups unth LAW4 and K-Selektnde
Substance m, K-Selektnde 3a-OH 3&OH 3a-OH 3/3-OH (%) b (%) b (46) b ($6) b Sa-ConfZguratum Sa-Androstan-17/?&3-one 12 88 88 12 Sa-Androstane-3,17-dtone 11 89 93 7 Sa-Estrane-3,17-dlone a 13 87 93 7 1-Methylene-5a-androstane_3,17dlone ’ 7 93 95 5 la-Methyl-5cr-androstan-17&ol-3-one 49 51 >99 Auabobc steroid Mam excreted substance Ollgm of Excretron parent and/or metabohte a substance used m urme c for confirmatton b Eolasterone 7a,l7a-Dnnethyl-5/3-androstane-3a,l7~-dtol(1) Synthesued ConJugated Boldenone Boldenone (II) Parent ConJugated 5/3-Androst-l-en-17El_ol3-one (III) Synthesued ConJugated cl-Cblorodehydro- 6~-Hydro~-4~hlor~deby~ometbyl- S~thes~d “Free” methyltestosterone testosterone Q ClosteboJ 4-~lofo-androst-4~en-3a~I-l7_one (VI Synthesued ~EIJU@@Sd Drostsnolone 2a-MethyI-5a-androstan-3a-ol-17-one @I) Syuthesmed &nJugated ~uo~terone 9a-~uo~l~nor-l7,17-d~ethyl- Syuthesrxed “Free” androsta-4,13-dlen-l~~~l-3~ne &iD 9a-Fluoro-l7a-methyl-aadrost-4-ene-3a,6~, “Free” 11~,17/3-tetrol wll~ Formebolone 2-Hy~~ethyl-l7a-me~l-andr~ta-l,4- Syuthesmed drene-lla,l7~-dml-3-one (TX) Ftlrazabol 16z-Hydro~r~a~l Unne ex study ConJugated Mestanolone 17a-Methyl-!5a-androstane-3a,t7~-~ol (X) Synthesmed cosrusa~ Mesterolone la-Methyl-Sa-androstan-3a-ol-17-one (XI) Synthestzed Comugated Metandrenone 17-Epunetandrenone (XII) Syuthesmed “Free” 6~-Hydro~et~dienone (XIII) Synthesmed “Free” 17a-Methyl-5~-sndrostane-3a,l7~-drol(XIV) Synthesmed CouJugated 17~-Methyl-S~-~~t-l-ene-3a,l7a~tol (XVI S~thes~ ConJugated Metlumdrtol 17a-Methyl-5#?-androstane-3a,l7~-drol (XIV) Synthestxed Comugated Metenolone Metenolone WI) Parent fzoqagated I-Methylene-5a-androstan-3a-al-17one BWD Synthesmed Comugated 17a-Metbyl-5a-audrostane-3a,l7~-dtol (X) Synthesrxed Conprgated 17a-Methyl-5jJ-androstane-3a,17/3-drol (XIV) Synthestxed co4sueated Nandrolone Sa-E&run-3a-o1-17-one (XVHD Synthesmed ckmJug&ted SjJ-Estran-3a-oL17-one (XIX) Synthesmed coslwmted Nore~~drolone 17a-~t~l-5~~s~ne-3a,l7~-dlol (XX) Synthesmed Cornugated Oxandroione Oxundroione (XXI) Parent ‘“Free” 17-Eptoxandrolone OfXU~ Synthesrxed “Free” Oxymesterone Oxymesterone WUII~ Parent CwJuBated Oxymetholone 17a-Meth~-Sa-~~~e-3a,l7~-dlol (X) Synthesixed CouJugated 2z-Hydroxymethyl-17a-methyl-5a-androstane- Unne ex study Comugated 3z,z,l7j%trtoJ 3’-Hydroxystanoxolol CCXIW Syuthesued ConJugated and “free” 3‘-Hydr~-l7~p~~~iol (XXVI Synthestxed “Free” 4B-Hydroxystanozoiol OCXW Synthesrxed Ck.mJugated 16&Hydroxystauozolol fXXVH) Synthesmed ConJugated a z = ~nfi~rat~n not rdenhfied b Urme ex study = metabohte obtamed from an excretron study c “Free” = un~nJugat~ W Schanzer and M Lbuke/Anal Chm~.Acta 275 (1993) 2348 29 Reductwn of 3-keto groups with K-Sekctnde tlon (Table 1) and it IS reduced m more than 85% The use of K-Selectnde [17,18] instead of yield to 3/3-hydroxy when the A-rmg has a 5/3 hthmm alummmm hydnde shows group selectrv- configuration (Table 11, m both mstances the lty, e g , the 3-keto group m 5a-estrane-3,17-dl- reaction yields the opposite configuration to re- one Hrlll be reduced first and then Hnth excess of duction mth hthmm alummnun hydnde When a reagent the 17-keto group reacts Moreover, the 3-keto-4-ene configuration IS reduced mth K- reduction of 3-keto groups 1s stereoselectlve, but Selektnde the 3-keto group reacts mamly to give different to hthmm alummmm hydnde reduction 3/I-hydrow smnlarly to the reaction wth hthmm The 3-keto group reacts m over 85% yield to @ve alummnun hydnde (Table 11, but the yield of the 3a-hydroxy when the A-ring has a 5a confqura- 3cy-isomer IS much higher than m the reduction TABLE 3 Temperature programme Koviits mdlces of anabohc steroids and theu metabohtes ’ Steroid C0lUmll A B C D E ov-1 ov-1 SE-54 SE-54 SE-54 Ollpm 033pm 025pm 025jkm 033pm Sa-E&ran-3a-o1-17-one bn-TMS @VIII) 2440 2453 2451 2454 2468 S/3-Androst-1-en-17/3-ol-3-one bB:TMS The results for column B (OV-1, 0 33 pm) and column C (SE-54, 0 25 pm) show for almost all the steroids almost Identical mdlces (dtierences less than 10 methylene umts) Oxandrolone (XXI) and eploxandrolone (XXII) both show a dtier- ence of about 30 methylene units and the metabohte of formebolone a dtierence of 16 methylene umts Ho&io& Mass spectrometry All anabohc steroids and their metabohtes were analysed as TMS denvafives by mass spec- trometry with EI lonlzatron at 70 eV General EI H H fragmentation patterns were regrstered for 17cu- Rg 1 Metabohsm of methyltestosterone (1) to 17a-methyl- methyl-17@hydroxy steroids and their 17-eplmers S~-androstane-3a,l7&dlol (X) and 17rr-methyl-Sp-andros- tane-3a,l7&dlol (XIV) which show as TMS derrvatlves an abundant D- rmg fragment at m/z 143 and Hrlth less mtenslty an ion at m/z 130 [5,23] and Segaloff m 1962 [61, ldenttfylng 17a-methyl- 16-Hydroxylated 17a-methyl-17/3-hydroxy ste- Sa-androstane-3cu,l7B_dlol (X) and 17a-methyl- roids display the correspondmg fragment ions 5/3-androstane-3a,l7B_dlol (XIV) as the mam (+ 88 u) when they are trnnethylsllylated, at m/z metabohtes (FG 0, a sun&u A-rmg metabohsm 231 and 218, respectively to that for testosterone [26]. 17-Keto steroids show a typical C/D-ring frag- Compared Hrlth the metabohsm of testos- ment at m/z 169 when the 17-keto group 1s terone, the ratio of the urmary excreted 17a- trunethylsllylated to an enol TMS derrvative The methyl 5a/5&lsomers 1s dflerent to the ratlo of fragment at m/z 169 IS SQII unexplamed and the androsterone to etmcholanolone (5a/5@, the only mformatlon 1s that deuteratlon at C-16 gave mam metabohtes of testosterone Excretion stud- a fragment at m/z 170 (results not shown) ies urlth a male volunteer showed a 5a/5/3 (X/XIX) ratio of 1 6 4 after oral apphcatlon of Metabohsm of anabol~c sterordr and synthesu of 100 mg of testosterone (O-60 h after apphcatlon) metabohtes and a ratio of 1 5 1 when 10 mg of methyltestos- The metabohsm of 20 anabohc steroids after terone were applied orally The same subject oral application was investigated (Table 2) For showed m both excretion studies an andros- the followmg dlscusslon the anabohc steroids were terone/etmcholanolone ratlo of 1 * 1 1 dlvlded accordmg to their structure mto five Both X and XIV were synthesized by a reac- classes androst-Cen3=ones, androsta-l&dren- tion of the Gngnard reagent methyhnagnesmm 3-ones, Sa-androstan-3-ones, Sa-androstanes wrth mdrde urlth ScY-androstan-3cY-o1-17-one (andros- special structure and methandnol terone) and 5@-androstan-3a-ol-17&one (etlo- For the mvestlgated anabohc steroids a brief cholanolone) The 17-keto group reacts 111about oversew of the mam excreted metabohtes and 95% yield to give a 17a-methyl-17&hydroxy theu synthesis IS presented structure The EI mass spectrum of X bs-TMS 1s shown Androst-4-en-3-on4zs III Fig 2 and that of XIV bls-TMS m Fig 3 Both Methyltestosterone Methyltestosterone (17u- derrvatlves show an abundant D-rmg fragment at methylandrost-4-en-17B_ol-3-one) was first syn- m/z 143 wrth 100% relative mtenslty (base peak) theslzed ml935 by Ruzleka et al 1241and m 1937 iVanddone Nandrolone (estr-4-en-17&o&3- by Oppenhauer E251 The metabohsm of methyl- one) was first synthesized m 1950 by Buch [27] testosterone m man was mvestigated by Rongone and by W&is and Nelson m 1953 [28] The 32 W Schanzer and M Lhmke /Ad Chm Acta 275 11993) 23-48 OH 1 O-TM CHa 80' 0 dP’ 143 B so- TM!+0 6 &H -2 no&Ho& H H Fig 4 Metabohsm of nandrokme (1) to Sa-estran-3a-al-17- one (XVIII) and Sp-estran-3a-oL17-one (XIX) I00 200 300 400 Mass/charge Fig 2 EI mass spectrum of 17a-methyl-Sa-andmstane- chrommm tnoxlde to yield estr+ene-3,17-dione 3a,17/3-d101bls-TMS (XI, molecular ion at m/z 450 Subsequent reduction with hydrogen m methanol using platmum dioxide as catalyst yielded 35% of 5ly-estrane-3,17-dione and 65% of S/3-estrane- metabolism was mvestigated by Engel et al m 3,17-dione Both isomers were separated by re- 1958 [71,who isolated two metabohtes Sa-estran- peated crystalhzatron and could be easily drstm- 3a=ol-17-one (XVIII) and 5~-estran-3a-ol-17-one gtushed by their meltmg pomts, which are 73- (KIK) (Fig 4) Both metabohtes were synthesized 75°C for 5a-estrane-3,17-dloneone[30] and 179- m 1960 by Kupfer et al [291 and m 1961 by 181°C for 5/3-estrane-3,17-d [31] Reduction CounseII [301 of 5a-androstane-3,17-dione with K-Selektrrde Both metabohtes were synthesixed here start- yielded XVIII m about 93% yield (Table 1) Re- mg with nortestosterone which was oxrdlzed with duction of 5/I-estrane-3,17-dlone with hydrogen using platmum dioxide as catalyst yielded about 64% of KIK and 36% of the S@somer, whereas the reduction with K-Selektride yielded KIK 111 143 / / about 6% and the 3/3-isomer m 94% yield (Table 0-TMS 1) c&l The EI mass spectrum of KVIII his-TMS is shown m Fig 5 and that of KIK his-TMS in Frg up 6 &3 TM!?-0 Norethundrohe Norethandrolone (17a-ethyl- &H estr-4-en-17/3-ol-3-one) was first synthesized m \ 1957 by Colton et aI [32] 17a-Ethyl-5a-estrane-3a,l7/3-diol and 17a- ethyl-5/3-estrane-3a,l7&diol (XI0 were reported as mam metabohtes by Brooks et al 181m 1971 When an excretion study was made of norethandrolone, only XX could be identified and no Sa-isomer of KX was detected (Fig 7) Both 300 400 h%/charge isomers were synthesized and are well separated Fig 3 EI mass spectrum of 17a-methyl-SB-androstane- m GC as his-TMS derivatives [difference of 40 3a,17/3dlol bls-TMS 0, molecular ton at m/z 450 methylene umts on an OV-1 cap&uy column W Schanzer and M Domk.e/Anal Chun Acta 275 (1993) 23-48 33 0-TMS 5 TMS-0 420 dPIi / HO $/pL;%$/pL_ H Fig 7 Metabohsm of norethandrolone (1) to 17a-ethyLS/3- estrane-3a,l7p-dlol (XX) and 17a-ethyl-5/3-estrane-3a,l7/3, 100 200 300 4L 2O-tno1(2) Masdchargs 1 Fq 5 EI mass spectrum of 5rr-estran-3a-ol-17-one bls-TMS (XVIII),molecular eon at m/z 420 mg 82% of 17a-ethyl-5/3-estrane-l7&ol-3-one, which was further reduced with hthmm alu- mmmm hydnde to 17a-ethyl-5&estrane-3a,l7/3- (column A, see Expenmental)] A further dlol (JQC) m 90% and the 3@lsomer m 10% yield metabohte was detected which 1s suggested to be (Table 1) the hydroxy metabohte of XX, hydroxylated at Figure 8 shows the EI mass spectrum of XX the 17a-ethyl group (Fig. 7) bs-TMS The spectrum shows no molecular ion The synthesis of XX started wth noreth- CM+, m/z 450) but the highest ion at m/z 435 androlone, which was reduced Hrlth hydrogen 111 (M+- 15) and 421 CM+- 29) Abundant frag- methanol-6 M sodmm hydromde (10 1, v/v) us- ments are the basic ion at m/z 157 and a frag- mg 10% palladmm on charcoal as catalyst, yield- ment Ion at m/z 144, both D-rmg fragments The 71 ?-TM i I5 7 I00 O-TM IB0 4/05 80 TM-0 CP ‘? H 20 f TMS-0 H ‘:’ 421 33 I / : 20’ 200 300 400 200 300 Hass/chargo 1 Masdchorgs Fq 6 EI mass spectrum of 5/I-estran-3a-al-17-one bu-TMS Ftg 8 EI mass spectrum of 17a-ethyl-5~-cstrane-3a,l7~~ol (XIX), molecular 10n at m/z 420 bs-TMS (XX), molecular Ion at m/z 450 34 W Schanzer and M Don& /AnaL Chmz.Actu 275 (1993) 23-48 ongm of these tons can be explained smularly to 143 / the D-rmg fragmentation of 17o-methyl-17/l-hy- I000 droxy trtmethylstlylated steroids (m/z 130 and 0-TMS 143, as shown m Figs 2 and 31, both ~wnh an addttional 14 u 800- Bolusterone Bolasterone (7a,l7ar-dtmethyl- e TMS-0 androst-4-en-17fi-ol-3-one) was synthestxed by 6 s00- P Campell and Babcock [33] There appears to be , no report of the metabohsm of bolasterone m 3 400 man I Excretion studtes wtth oral adnumstratton of 20 mg of bolasterone to two male volunteers show two metabohtes excreted conjugated mto urme Both metabohtes show a molecular ion 4 u hrgher than bolasterone Synthesis of 7cr,17 OH ‘h 1 A-4 0 ’ 0 ’ @ Cl 43”bH Fig 13 Structure of oxymesterone (XXIII) I 0 V &y?nestero~ &XJ..Z) Oxymesterone (XXIII) / (17a-methylandrost-4-ene-4,17~-drol-3-one, Frg HO @ 131, whrch was synthesrzed m 1965 by Camenno Cl 1381,IS less metabohzed and mamly excreted un- Ftg 11 Metabohsm of clostebol (1) to 4-cbloroandrost-4-en- 3a-ol-17-one 0 changed as a comugate The hydroxy group at C-4 may hmder the reductron of the 4,5-double bond and/or allow a very raped conlugatron of tent and the 3a-hydroxydehydrogenase can re- XXIII at the 4-hydroxy group and excretron mto duce clostebol-17-one m the presence of the 4,5- ufllle double bond to yield V, which 1s coaugated and The EI mass spectrum of XXIII tns-TMS (Frg excreted as the mam metabolite 14) IS dommated by an abundant molecular ion at The synthesis of V started mth clostebol, which m/z 534 was oxrdrxed wrth chronuum trroxrde to 4-chloro- Fhoxymesterone Fluoxymesterone (9a-fluoro- androst4ene-3,17-dione foknved by reduction 17a-methylandrost-4-ene-11&17&drol-3-one) was wrth K-Selektnde to yield a nnxture of 19% of V synthesrxed m 1956 by Herr et aI [39] In 1990 and 81% of the correspondmg 3&isomer (Table Kammerer et al [40] published a GC-MS mvestr- 1) gatron of fluoxymesterone metabohsm m man The EI mass spectrum of V bts-TMS 1s shown They excluded excretion of A-nng reduced m Ftg 12 metabohtes by usmg reference substances o-TM3 100 CHa O-TM!3 88 TM-O @ O-TMS 6ft 60 40 20 0 j 0I, 100 280 380 400 200 300 400 500 Mass/charge Moss/charge Fw 12 EI mass spectrum of 4-cbloroandrost4en-3a-oI-17- Fm 14 EI mass spectrum of oxymesterone tns-TMS (XXIII), one bwTMS (v), molecular Ion at m/s 466 molecular Ion at m/t 534 36 W SchZnzer and M Lkmh /Ad Chm Acta 275 (1993) 2348 OH CHs 00- ;2 8 I J‘X 6 60. CH3 e3 2OB 9 / J 40. 357 ‘/“I 342 \ 372 447 I CryI-l _I / , ‘, 4 100 200 380 400 Mass/charge Fig 15 Metabolism of fluoxymesterone (11, 6jLhydroxy- FU 16 EI mass spectrum of 9a-fluoro-l&nor-17,17-dlmethyl- fluoxymesterone (21, 9a-flu0ro-18-n0r-17,17-dunethy1-4,13& 4,13-d~ene-ll/?-ol-3+cmebe-TMS (VII), molecular ion at m/z ene-ll,%ol-3-one (VII) and 9a-fluoro-17a-methylandrost-4- 462 ene-3a,6~,11~,17~-tetrol (VIII) MSTFA-1m1 (100 1, v/w) 1s unsatisfactory This The present mvestigatlons confiied 9a-flu- should be consldered when fluoxymesterone oro-l&nor-17,17-dunethyl-4,13-drene-11/3-ol-3- metabohtes are screened m the uncoqugated one (VII), 6/3-hydroxyfluoxymesterone and 9a- urme fraction fluoro-17a-methylandrost-4-ene-3(w,6/3,11/3,17/3- tetrol (VIII) as the mam metabohtes (Fig 15) All three metabohtes were synthesized A detailed pubhcatlon 1s m preparation Compound MI was 1 73 obtamed by refluxmg fluoxymesterone m acetom- 1000 -1i trde-trlfluoroacetlc acid (10 1, v/v) 6p-Hy- 0-TMS droxyfluoxymesterone was synthesized via direct CH3 oxldatlon of the enol-TMS derivative of flu- 908i oxymesterone m ethanol catalysed by sunlight The reduction of 6/3-hydroqfluoxymesterone with e6 600- K-Selektnde yielded VIII m about 72% and the 0 0-TMS 3@lsomer m 28% yield (Table 1) 5 2 The EI mass spectrum of VII bls-TMS 1s shown 400- m Fig 16 and that of VIII tetra-TMS m Fig 17 The derlvatlzatlon of VII and VIII 1s satlsfac- tory with MSTFA-ammomum lodlde as de- scribed above, VII reacts wlthm 5 mm at 80°C completely to a bls-TMS derivative whereas I00 200 300 400 500 600 trlmethylsllylatlon of the 11/l-hydroxy group m Mass/charge VIII 1s sterlcally hmdered by the fluorme atom at Fig 17 El mass spectrum of 9a-fluoro-17a-methylandrost-4- C-9a and the reactlon needs 2 h at 8O”C, yleldmg ene-3ru,6&11&17&tetrol tetra-TMS (VIII), molecular Ion at a tetra-TMS derlvatlve Derlvatlzatlon with m/z 642 W S~~~IUU and M Lhwuke/Atd Chum Acta 275 (1993) 23-48 37 0-TMS 430 \ 1 380 400 Mass/charge Fig 19 EI mass spectrum of boldenone bts-TMS (II), molecu- lar Ion at m/z 430 Fa 18 Metabolwm oi boldenone (II) to $hmhst-1-en-17& ol-3-one (III), androsta-l+dlene-3,17-dlone (11, J&androst- Metandmone Metandlenone (17ar-methyl- lent-3,17&one (21, 5p-androst-l-ene-3a,l7&dlol (3) and androsta-1,4-dlen-17/3-ol-3-one) was synthesized 5j?-androst-l-en-3a-o1-17-one (4) 1111955 by Vlscher et al [421 by rmcroblologtcal dehydrogenatlon of methyltestosterone In 1956 Meystere et al [41] reported the dehydrogenatlon Androsta-1,4-a!m-3-ones of methyltestosterone vvlth selenium dloxlde 68- Androsta-1,Cdlen3-ones are reduced by the Hydroxymetandlenone (XIlI) was identified m S@reductase only, but to a smaller extent than 1963 by Rongone and Segaloff as the mam testosterone. In this mstance 6p-hydroxylatlon 1s metabohte [4], which was excreted uncoqugated favoured for metandlenone, 4-&lorodehydro- m urme A further metabohte 1s 17-eplmetan- methyltestosterone and fluoxymesterone Bokhone Boldenone (II) (androsta-1,4-dlen- 17@ol-3-one) was synthesized 1111956 by Meystre 194 et al [41] / The metabolism of II was mvestlgated m 1971 100- 0-THS by Galettl and Garde [l] GC-MS of boldenone and its mam metabohtes was reported by SB- Schanzer and Doruke m 1992 [12] Boldenone itself 1s excreted as a coqugate m urme The mam metabohtes are S/3-androst-l-en- 17/3-ol-3-one (III), S/3-androst-l-en-3cr-ol-17-one and 5/3-androst-l-ene-3a,17/3-&ol (Fig 18) In the proposed screenmg procedure II and III are used to detect boldenone rmsuse The synthesis of III started wth II, which was selectively reduced Hrlth hydrogen m methanol- potassium hydroxide using 10% palladium on NB 300 400 charcoal as catalyst [12] Mass/charge The El mass spectrum of II bls-TMS 1s shown Fu 20 EI mass spectrum of Qhndrost-1-en-17&ol-3-one m Fig 19 and that of III bls-TMS m Fig 20. bu-TM.5 (III), molecular Ion at m/z 432 38 W Schanzerand iU Don&e /Anal Chun Acta 275 @93) 23-48 .@+_ $/y% o@sH;$ 0 H Fig 21 Metabohsm of metandlenone (1) to l’l-eplmetandlenone (XII), 6p-hydroxymetandlenone (XIII), 17u-methyK+ndros- tane-3a,l7&dlol (XIV) and 17~-methyl-5~-androst-l-ene-3a,l7~-d~ol (xv) drenone (XII) obtamed from the uncomugated 3 and XV bn-TMS m Ftg 24 All these EI mass fraction which was tdenttfred and synthesized m spectra are donunated by the D-rmg fragment at 1971 by Macdonald et al [S] In 1980 Durbeck m/z 143 and Bucker [231 mvestlgated the uncomugated 4-Chlorodehydromethyltestosterone. 4-Chlom- urme fractton after application of metandrenone dehydromethyltestosterone Gt-chloro-17a-methyl- by GC-MS and discussed the EI fragmentatron androsta-l,4-dlen-l7/3-ol-3-one) was synthesrxed of the trrmethylsrlylated metabohtes In 1991 m 1960 by Schubert et al [43] The metabohsm of Schanxer et al [13] pubhshed the tdentlficatron of 4-chlorodehydromethyltestosterone m man was A-rmg reduced metabohtes excreted as comu- reported by Schubert and co-workers [2,3] In gates m urme, 17a+methyl-5/3-androst-l-ene- addition to the excretron of Cchlorodehydro- 3a,17#I-drol, tts 17-eprmer 17/3-methyl+ methyltestosterone &elf they also tdentrfied 6/3- androst-1-ene-3cu,l7o-drol (xv) and 17a-methyl- hydroxy+chlorodehydromethyltestosterone (IV), 5/3-androstane-3a,l7/3-&ol (XIV) (Fig 21); XIV 1s 16#&hydroxy-4-chlorodehydromethyltestosterone also a metabohte of methyltestosterone and methandrtol The syntheses of 6/3-hydroxymetandtenone (XIII) was performed as descrrbed above by au- tooxtdatron of the enol-TMS dertvattve m ethanol catalysed by sunlight The synthesis of XII was performed vra the 17@sulphate of metandtenone whrch spontaneously decomposed m water gtvmg several dehydration products and XII 111about 0 30% yreld Syntheses of XV started wrth 17-epr- metandrenone (XII), whtch was selectrvely re- duced with hydrogen m methanol-potassium hy- 282 droxtde using 10% palladium on charcoal grv- \ mg 17/3-methyl-5~-androst-l-en-l7~-ol-3-one m about 20% yteld The reductton of this 3-ketone wtth hthmm ahummum hydrtde gave XV m more than 85% yteld For the syntheses of XIV, see the 0 I0B em 300 dtscusslon of methyltestosterone Mass/charge EI mass spectra are shown for XII TMS m Fig Fig 22 EI mass spectrum of 1Fepunetandlenone TMS (XII), 22, XIII ba-TMS m Ftg 23, XIV bls-TMS m Fig molecular ion at m/z 372 39 143 / TYS-0 H 350 / / 216 440 268 343 i \ Mass/charge 208 306 Mass/charge Fu 24 EI mass spectrum of 17&methyl-5&androst-l-ene- Fig 23 EI mass spectrum of 6/3-hydroxymetandlenone bls- 3a,17a-dlol bwTTvlS (XV), molecular 1011at m/z 448 Thm (ran), molemllar 1on at m/z 460 same El mass spectrum as the parent sterord and and 6p,16p-dlhydroxy-4-chlorodehydromethyl- It was therefore assumed to be a 17-epnner They testosterone as the mam metabohtes In 1983 further recorded a brshydroxy metabohte whrch Durbeclc et al [44] studled the metabohsm of was consrdered to be a 6/3,12-drhydroxy metabo- 4-chIorodehydromethyltestosterone by Gc-MS hte (Fig 25) They were able to txmfirm6/3-hydroxy4-chloro- In an excretion study wrth 22 mg of orally dehydmmethyltestosterone ow and the 6/3,16/3- admmtstered 4-&lorodehydromethyltestosterone, dlhydroxy metabohte They dld not detect cchlo- it was possible to rdentrfy m the unconmgated rodehydromethyltestosterone and the 16&hy- urme fractron a 17-eplmer, whrch was synthesrxed droxy metabohte, but a substance showmg the [15], 6/3-hydroxy-4chlorodehydromethyltestos- Cl OH Fig 25 Metabohsm of 44lorodehydromethyltestosterone (1) to 6&hydroxy4&lorodehydromethyltestosterone (IV), 6&16p-dlhy- droxy4chlorodehydromethyltestosterone (2) and 6&12z-d~ydroxy4chloradehydromethyltestostestostene(3) (z = umdent&d con- &ration) 40 W Schanzer and M Do&e /Anal Chm Acta 275 (1993) 23-48 terone (IV), which was also synthesized, a 6/3,16- OH dlhydroxy metabohte and a 6/3,12-dlhydroxy metabohte, as dlscussed by Durbeck et al 1441 The 12-hydroxy group could be confirmed usmg the EI mass spectra of the tns-TMS derivative of this metabohte, drsplaymg an abundant fragment at m/z 170 This fragment was previously re- ported [13] to orlgmate from a 12,17-dlhydroxy- 17-methyl-bls-TMS structure Compound IV was synthesrzed by the above- described synthesis The enol-TMS ether was au- tooxldrzed when dissolved m lsopropanol and ex- posed to sunlight, yleldmg IV A detailed descrlp- Fig 27 Metabohsm of formebolone (1) to 2-hydroxymethyl- 17a-methylandrosta-l,4-dlene-1la,l7~-d~ol-3-one (IX) tron of this synthesis 1s m preparation The EI mass spectrum of IV bls-TMS 1s shown m Fig 26 The D-rmg fragment at m/z 143 1s the In the present mvestlgatlon wrth 20 mg of most abundant ion The fragments at m/z 243 formebolone applied orally to a male volunteer, a and 315 are the 4-chloro analogue fragments of reduced metabohte was identified 111the unconlu- the ions at m/z 209 and 281 of 6&hydroxy- gated fraction (Fig 27) after basic extractlon metandlenone bls-TMS (Fig 23) This polar metabohte was ldentlfied by synthesis Formebolone Formebolone (2-formyl-17a- as 2-hydroxymethyl-17a-methylandrosta-1,4-dl- methylandrosta-1,4-dlene-lla,l7&d~ol-3-one) ene-lla,l7/3-dlol-3-one (IX) was synthesized m 1965 by Canomca et al [451 Compound IX was synthesized by reduction of A GC-MS mvestlgatlon of formebolone formebolone wrth K-Selecttrde m tetrahydrofu- metabolism m man was published by Masse et al ran The K-Selectnde showed a selectnqty to the wd formyl group which was first reduced and the 3-keto group remamed mtact when the reagent was not used m excess Isolation of IX by prepar- O-TM ative HPLC welded a pure reference substance CHa The EI mass spectrum of IX tns-TMS 1s dls- 143 played m Fig 28 \ 100- 7 Sa-Androstan-3-ones Cl 0-THS Drostanolone Drostanolone (2a-methylJa- 80- androstan-17/3-ol-3-one) was synthesized m 1959 315 by Rmgold et al [47] Drostanolone 1s orally apphed as proplonate After oral apphcatlon of 20 mg of drostanolone 243 proplonate to a male volunteer, 2a-methylJa- / androstan-3a-ol-17-one (VI) was ldenttied as the 217 mam metabohte (Fig 29), which IS excreted as a 347 2p5 I conjugate m urme No drostanolone or dro- 404 stanolone proplonate was excreted / yg_494 The synthesis of VI started with drostanolone, 0 L A,iL., 100 200 300 400 500 which was oxldlzed wth chrommm tnoxlde to Moss/charge 2a-methylJa-androstane3,17-dlone and then re- Fq 26 EI mass spectrum of S/3-hydroxy-4-chlorodehydro- duced with K-Selehde yleldmg about 99% of methyltestosterone bwTMS (IV), molecular ion at m/z 494 2a-methylJa-androstan3a-ol-17-one (VI) and W Schc”uutrand M Domke/AnaL Ch Acta 275 (1993) 23-48 100. O-T6 Ma CHa &$FTS 80. TUS-0 w H I3 68 60- z 60 P 6 P 3 253 40. 9 40 I T 20 358 0 lL 100 200 300 400 500 Mass/charge Mass/charge Fu 28 EI mass spectrum of 2-hydroxymethyl-17a-methyl- Fw 30 EI mass spectrum of 2a-methyl-5a-androstan-3a-al- androsta-1,4d~ene-lla,l7~-&ol-3-one tns-TMS (IX), molecu- 17-one ba+TMS (VI), molecular 10x1at m/r 448 lar 1011at m/z 562 The synthesis of XI was camed out m the 1% of the 3@somer (Table 1) same way as that of the drostanolone metabohte The EI mass spectrum of VI bls-TMS is pre- VI Startmg mth the chrommm tlroxlde oxldatlon sented m FM 30 of mesterolone la-methyl-5a-androstane-3,17-dl- Mesterolone Mesterolone (la-methyl&-an- one was obtamed, which was reduced wth K- drostan-17/3-ol-3-one was synthesized by Wiechert Selektrlde to 99% of 1a-methyl-Sa-androstan-3a- Ml ol-17-one (XI) and 1% of the 3@somer (Table It 1s metabolized 111 the same way as 1) drostanolone After oral apphcatlon of 20 mg of The EI mass spectrum of XI bls-TMS 1s shown mesterolone the mam metabohte was ldenttied m Fig 32 as la-methyl-5a-androstan-3a-o1-17-one (XI), ex- Mestunohe Mestanolone (17a-methylJa- creted cowugated m urme (Fig 31) No parent androstan-17/3-c&3-one) was synthesized m 1935 drug was detected by Ruzlecka et al 1241 OH OH 1 1 ‘% C&t 0 dP 0 H &H 1 0 1 q VI XI CHa CHa HO dP HO H c@H Fu 29 Metabohsm of drostaoolone (1) to 2a-methyl&- Fu 31 Metabohsm of mesterolone (1) to la-methylJa- androstan-3a-oL17-one (VI) androstan-3a-o1-17-one (XI) 42 W Schanzcr and M Don& /Am! Chtm. Acta 275 @!?3) 23-48 OH 73 \ XVI CHa 108 II-TMS / BB 0 dPH 4s 0 8 60 1 6 -a XVII CH2 s 40 AJ$J HO w H 20 F@ 34 Metabolism of metenolone (XVI) to 1-methylen&- androstan-3a-oL17-one (XVII) 9 0; 100 200 300 Mass/charge m 1960 by Wlechert and Kaspar [49] Metenolone F@ 32 EI mass spectrum of la-methyl-5a-androstan-3a-ol- 17-one bls-TMS (XI), molecular Ion at m/z 448 IS applied orally as the acetate or by mtramolecu- lar mjectlon as the enanthate The metabohsm of XVI was investigated by Gerhards et al m 1965 After oral admmlstration of 20 mg of mes- [501, ldentlfylng l-methylenJa-androstan3a-ol- tanolone to a male volunteer, 17a-methyl-5a- 17-one (XVII) as the mam metabohte and un- androstane-3a,l7&dol (X) was Identified as the changed metenolone (XVI), both excreted as con- mam metabohte, excreted as a coqugate m urme jugates (Fig. 34) Recently Goudreault and Masse (Fig 33) No parent drug was detected The same [51] descriid a GC-MS mvesfigation of XVI and metabohte was obtamed m the metabolism of its metabohtes methyltestosterone and oxymetholone In this work the synthesis of XYII started wth For the synthesis of X see the discussion of metenolone (XVI), whch was omtid to l- methyltestosterone and for the EI mass spectrum methyl-5a-androst-1-ene-3,17-dlone The latter of X bls-TMS see Fig 2 was lsomenzed to l-methylen-5a-androstane- Metendone (i#W.. Metenolone (l-methylJa- 3,17-dlone as described by Wlechert et al [52] androst-1-en-17/3&3_one) (XVI) was synthesized usmg the method of Rmgold and Malhotra 1531, who converted a&unsaturated ketones mto p,-y-unsaturated ketones with potassium tert- butoxlde m tert-butanol OH CH, The reduction of the lsomerued product Wlfh K-Selektnde yielded 80% of XVII, 10% of the correspondmg 3@somer and about 10% of l- methyl-5a-androst-1-ene-3/3,17/3-dlol (Table 1) The EI mass spectrum of XVI bls-TMS 1s presented m Fig 35 and that of XVII be-TMS m 1 OH Rg 36 X CHs Oxymethokwze Oxymetholone (Zhydroxymeth- ylene-17a-methylJa-androstan-17/3-ol-3-one) was f-l@ first prepared by Rmgold et al m 1959 [47] Oxymetholone 1s substantially metabohzed and Fig 33 Metabohn of mestanolone (1) to 17a-methyLSa- several metabohtes can be detected by GC-MS androstane-3a,l7p-dlool(X) In the neutral and basic fraction used for screen- W Schanzcrand M Lbtuke/AnaLChun Acta 275 (1993)23-48 43 195 OH / O-TMS 1 CHa CHZ HO-CH I zcf!P TMS-0 &H 208 I / 0 Ii, , , .A Fig 37 Metabohsm of oxymetholone (1) to 17a-methyLSa- I00 2110 300 488 androstane-3a,l7&dlol (X) and 2z-hydroxymethyl-17a- Mass/charge methyl-5a-androstane-3z,z,l7p-trtol (2) (z = umdentlfied FQJ 35 EI mass spectrum of metenolone bls-TMS (XVI), configuration) molecular ion at m/z 446 group yields X with a 3a-hydroxy configuration mg of anabohc steroids, 17a-methyl&-andros- A further metabohte IS detected m excretion tane-3cu,l7/3-dlol (X1 was ldentdied as a metabo- studies with oxymetholone Hrlth a molecular Ion hte The ongm of this metabohte can be ex- at m/z 640, which 1s explamed as a hydroxy plamed by orndatlon of the 2-hydroxymethylene denvatlve of the completely reduced oxymeth- group to a 2-carbomc acid This oxidation prod- olone (F& 37) This metabohte 1s further used uct IS a /3-ketocarbomc acrd, which is less stable for screenmg of an oxymetholone abuse even and decomposes to 17a-methyl&-androstan- though rts structure 1s not completely known 17/3-ol-3-one Subsequent reduction of the 3-keto The synthesis of X 1s described m the discus- slon of methyltestosterone and the EI mass spec- trum of X bn-TMS 1s shown m Fig 2 1‘2 O-TM Sa-Androstanes wrth specs& structure CH2 Oxandrolone (%X7) Oxandrolone (XXI) (17a- methyl-2-oxa-Sa-androstan-17/3-ol-3-one) was synthesized m 1962 by Pappo and Jung [54] In TMS-0 1989 Masse et al 1551published a GC-MS mves- OPH 44 43< / tlgatlon of oxandrolone metabolism m man Com- pound XXI 1s excreted unchanged to a great extent As the mam metabohte a 17-eplmer (XXII) of oxandrolone (XXI) was described (Fig 38) Further metabohtes hydroxylated at C-16 were detected by GC-MS but m low concentration In this work XXII was synthesized by the above described method for 17-epunerzatlon [ 151 0 50 I00 158 208 250 300 358 400 450 The E&mass spectra of XXI TMS m Fig 39 Moss/charge and XXII TMS m Fig 40 show that these spectra Fu 36 EI mass spectrum of l-methylen-5a-androstan-3a_ol- are identical and dominated by the abundant 17-one bls-TMS (XVII), molecular eon at m/z 446 D-rmg fragment at m/z 143 W Scharuer and iU Do& /AnaL Chm Acta 275 (1993) 23-48 OH / - 100- O H Fig 38 Metabolism of oxandrolone (XXI) to 17-epmxandro- lone (XXII) 200 Furazubol Furazabol was synthesized m 1965 Moss/charge by Ohta et al [56] Metabohsm studies of furaza- F@ 40 El mass spectrum of 17-epmxandrolone TMS (XXII), molecular Ion at m /z 378 bol vvlth rats were published by Takegoshl et al 1571,but no study of the metabohsm of furazabol 111man has been reported Because no nususe of furazabol has been re- After oral admmlstratlon of 20 mg of furazabol ported by any IOC accredited laboratory, this m man furazabol itself and a metabohte hydroxyl- metabohte was not synthesned but a reference ated at C-16 were detected m the coqugated obtamed from an excretion study mth furazabol urme fraction (Fig 41) The EI mass spectrum of was used for screemng purposes the TMS derlvatrve of this metabohte m Fig 42 Stanozolol Stanozolol (17a-methyl-17@hy- show fragments at m/z 218 and 231, confirming droxy-5a-androst-2-eno[3,2-cjpyrazole) was syn- a D-rmg wth a 17-methyl-16,17-dlhydroxy-bls- theslzed m 1959 by Clmton and co-workers TMS structure [58,59] The metabolism of Stan020101in man was investigated m this laboratory and pubhshed m 1990 [14] About eleven metabolites were confiied and the mam metabohtes were synthesized, namely 40 0 100 200 300 400 Mass/charge F@ 39 EI mass spectrum of oxandrolone TMS (XXI), molcc- Fu 41 Metabohsm of furazabol(1) to 16z-hydmbol ular eon at m/r 378 (2) (.z = umdenttied configuration) W Schanzer and M Lbmke/Ana& Chun Acta 275 (1993) 23-48 45 O-TM5 100- !31 ma 3 0-TMS 1 LiN 60. k’ & 490 e H \ s 60 0 5 3 40 545 I 560 / 20- ‘:’ / 455 59 0 11 1 I,, 400 300 500 100 200 300 400 500 Moss/charge Moss/charge FIN 42 EI mass spectrum of 16z-hydroxyfurazabol bls_TMS F@ 44 EI mass spectrum of 3’-hydroxystanozolol tns-TMS (z = umdenttied configuration), molecular ton at m/z 490 OQClV),molecular Ion at m/z 560 3’-hydroxystanozolol (XXIV), 3’-hydroxy-lir-epr- Ruzrecka et al. [241. It 1s apphed orally as the stanozolol (XXVI 1151, 4&hydroxystanozolol drpropronate i/I and 168_hydroxystanoz,olol (XXVII) (Fig The metabohsm of methandnol and meth- andnol drpropronate was mvestrgated and after EI mass spectra are displayed for XXIV tns- 30 mg of orally apphed methandnol drproptonate TMS m Frg 44, XXV tns-TMS 111Frg. 45, XXVI excreted methandnol was rdentrfied 111very low trrs-TMS m Frg 46 and XXVII tns-TMS m Frg concentratron m the sulphate fraction which 1s 47 routmely not controlled The mam metabohte Mef/rundrwL Methandnol (17a-methylandrost- was rdentrfied as 17a-methyl-5@androstane- !I-ene-3/3,17&drol) was synthesrzed m 1935 by 3a,17&drol (XIV) (Fig 48) 111the coqugated unne H- F@ 43 Metabobsm of stanuzolol (1) to 3’-hydroxystanozolol @.XW, 3’-hydroxy-17-eprstanozolol (XXVI) and 16@-hydro~tanozolol orrwn) 46 W Schanzer and M Dotuke /And Chm. Acta 275 (1993) 23-48 73 / 100' 73 : 80- -3:! 60- B 231 s / 9 40. 5/60 i 38 I 20 ‘i” ,53/ 470 ’ 20- 26 I / / I 0 ll,l*, ‘.I!\ 01 100 200 300 400 500 100 Mass/chary Mass/charge Wg 47 EI mass spectrum of 168.hydroxystanozololtns-TMS Fig 45 3EI mass spectrum of 3’-hydroxy-17-epwanozolol tns- @XVII), molecular ion at m/z 560 TMS (XXVI,molecu\ar Ion at m / 2 560 C-4,5, sumlarly as the metabohsm of dehy- fraction, but m low concentration An excretion droepumdrosterone (androst-5-en-3/3-ol-l7-one). study with 20 mg of methandnol confirmed The oxldatlon and lsometrzatlon product 1smeth- metabohte XIV as the mam metabohte and a yltestosterone followmg the above-described possible Sa-Isomer was not detected metabohc pathway v&h the excretion of mamly Thus result can only be explamed by a metabohc XIV, a metabohte with a 3a-hydroxy cotigura- pathway of methandnol m man mcludmg 3-oxlda- tion tlon and lsomenzation of the 5,ddouble bond to For the synthesis of XlV see the discussion of methyltestosterone and for the EI mass spectrum of XIV bls-TMS see Fig 3. P-TMS CH3 The authors are grateful to the Bundesmstltut of Spomssenschaft, Cologne, and the Interna- TMS- OH CHI Ll7 I 0 11,. .I!. I00 200 :c!o 4L10 W0 Mass/charge Fu 46 EI mass spectrum of 4fi-hydroxystanozololtns-TMS Fig 48 Metabohsm of methandnol (1) to 17cr-methyl-5/3- (XXVI),molecular Ion at m/z 560 androstane-3a,l7&dol (XIV) W Schanzer and A4 Don&e /Anal Chm Acta 275 (1993) 23-48 47 tlonal Athletlc Foundatron for financml support 24 L. Ruxu~ka, A4W Goldberg and H R Rosenberg, Hetv They are also mdebted to Dr H Geyer for Chun Acta, 18 (1935) 1487 25 RV Oppenauer, Reel Trav Chum Pays-Bas, 56 (1937) asswtmg IIIexcretion studies 137 26 C D Kochakmn, Stemds, 55 (1990) 92 27 kJ Buch, J Chem Sot, (1950) 367 28 AL Wilds and N A Nelson, J Am Chem Sot ,75 (1953) REFERENCES 5366 29 D Kupfer, E Forchlelh and R I Dorfmann, J Org Chem , F Galettl and R Garde, Steroids, 18 (1971) 39 25 (1960) 1674 K. Schubert and K Wehrberger, Endokrmolo@e, 55 (1970) 30 R E Counsell, Tetrahedron, 15 (1961) 202 257 31 R T Rapalla and E Furkas, J Am Chem Sot ,80 (1958) K. Schubert and G Schumann, Endokrmolo@e, 56 (1970) 1008 172 32 F B C&on, L N Nysted, B megel and AL Raymond, J E L Rongone and A Segaloff, Steroids, 1 (1963) 179 Am Chem Sac , 79 (1957) 1123 B S Macdonald, P J Sykes, PM Adhdcary and RA 33 J A Campbell and J C Babcock, m L Martin1 and A Harkness, Steroids, 18 (1971) 753 Pecde (Eds ), Hormonal Steroids, Proceedmgs of 1st Inter- E L Rongone and A. Segaloff, J BIOI Chem , 237 (1962) national Congress, Vol 2, Acadenuc Press, New York, 1066 1965, p 59 L L Engel, J Alexander and M Wheeler, J BIOI Chem , 34 B Camermo, B Patelh, A Vercellone and F Meda, 231 (1958) 159 Farmaco, Ed SCI , 11 (1956) 586 C J W Brooks, AR Thawley, P Rocher, B S Mlddle- 35 B Camermo, B Patelh and A Vercellone, J Am Chem ditch, G M Anthony and W G Stdlwell, J Chromatogr Sot , 78 (1956) 3540 SC1 ,9 (1971) 35 36 H J Rmgold, E Batres, 0 Mancera and G Rosenkranz, 9 M Don&e, J Zmnnermann, K. R Barwald, W Schanzer, J Org Chem ,21(1956) 1432 V Chnst, K Klostemmnn and G Opfermann, Dtsch Z 37 L Starka, L Sickmann, H D Hoppen and H Breuer, Sportmed ,35 (1984) 14 Arznelm -Forsch , 19 (1969) 2022 10 M Domke, H Geyer, A. Gotxmann, M Kraft, F Mandel, 38 B Camenno, m L Martml and A. Pecde (Eds ), Hor- E Nolteemstmg, G Opfermann, G Sigmund, W Schanzer monal Steroids, Proceedings of 1st International Congress, and J Zlmmermann, m P Bellott], G Benxr and A Vol 2, Academic Press, New York, 1965, p 51 LJungqwst (Eds ), Official Proceedmgs, InternatIonal Ath- 39 ME Herr,JA HoggandRH Levm,J Am Chem Sot, letlc Foundation World Symposmm on Dopmg m Sport, 78 (1956) 501 Florence, Intematlonal Athletic Foundation, Monaco, 40 R C Kanunerer, J L Merdmk, M Jagels, D H Catlm and 1988, p 53 KX. Hul, J Steroid Blochem, 36 (1990) 659 11 R Masse, C Ayotte and R Dugal, J Chromatogr , 489 41 C Meystre, H Frey, W Voser and A Wettstem, Helv (1989) 23 Chun Acta, 39 (1956) 734 12 W Schanzer and M Domke, B~ol Mass Spectrom, 21 42 E Vlscher, C Meystre and A Wettstem, Helv Chum (1992) 3 Acta, 38 (1955) 1502 13 W Schanzer, H Geyer and M Domke, J Steroid Blochem 43 A Schubert, A Stachowmk, D Onken, H Specht, K Mol Blol , 38 (1991) 441 Barn&o]-Oettler, E Bode, K Heller, W Pohnert, S 14 W Schanzer, G Opfermann and M Domke, J Steroid Schwarz and R Zepter, Pharmaue, 18 (1963) 323 Bmchem, 36 (1990) 153 44 H W Durbeck, I Buker, B Scheulen and B Tehn, J 15 W Schanzer, G Opfermann and M Domke, Steroids, 57 Cbromatogr SCI , 21 (1983) 405 (1992) 537 45 L Canomca, G Jomnu, F Pelhzom and C Scolastlco, 16 M Do&e and 3 Zmnnermann, J Chromatogr ,202 (1980) Gazz Chum Ital, 95 (1965) 138 483 46 R Masse, H BI and P Du, Anal Chum Acta, 247 (1991) 17 CA Brown, J Am Chem Sot, 95 (1973) 4100 211 18 B Ganem, J Org Chem ,40 (1975) 146 47 H J Rmgold, E Batres, 0 Halpem and E Coechea, J 19 S Burstem, R I Dorfman and E M Nadel, Arch Bmchem Am Chem Sac , 81(1959) 427 Bropbys , 53 (1954) 307 48 R Wiechert, m L Martun and A Pecde (Eds 1, Hormonal 20 EM Nadel, S Burstem and R I Dorfman, Arch Bmchem Steroids, Proceedings of 1st Intematlonal Congress, Vol Blophys , 61 (1956) 144 2, Acadenuc Press, New York, 1965, p 77 21 R Garde and A Lusignam, J Org Chem, 32 (1967) 2647 49 R Wlechert and E Kaspar, Chem Ber , 93 (1960) 1790 22 P 0 Edlund, L Bowers and J Hemon, J Chromatogr , 50 E Gerhards, KH Kolb and P E Schulze, Hoppe Seylers 487 (1989) 341 Z Pbyslol Chem , 342 (1965) 40 23 H W Durbeck and I Bucker, Boomed Mass Spectrom , 51 D Goudreault and R Masse, J Steroid Bmchem Mol 700) (1980) 437 Blol , 37 (1990) 137 W Schanzu and hi Dontke/AnaL Chm Acta 275 (1993) 23-48 52 R Wnxhert, U Kerb and P E Schulze, Natunws- 57 T Takegoslu, H Tachlzawa and G Ohta, Chem Pharm senschaften, Sl(1964) 86 Bull, 20 (1972) 1243 53 H J Rmgold and S IL Malhotra, Tetrahedron L&t, (1962) 58 R 0 Clmton, A J Manaon, F W Stonner, A.L Beyler, 669 G 0 Potts and A Arnold, J Am Chem Sot, 81 (1959) 54 R Pappo and C J Jung, Tetrahedron Lctt., (1962) 365 1513 55 R Masse, H h, C Ayotte and R Dugal, Blamed Envl- 59 R 0 Chnton, A.J Manson, F W Stonner, H C Neumann, ran Mass Spectrom ,18 (1989) 429 R G Chrtstransen, R L Clarke, 3 H Ackerman, D F Page, 56 G Ohta, T Takegoshl, K. Ueno and M Shnmzu, Chem J W Dean, W B Dlckmson and C Carabateas, J Am Phann Bull, 13 (1965) 1445 Chem Sot , 83 (1961) 1478